AIDS title: Design of Antiviral Drugs Effective Against the HIV-1 VirusDuring the last year our work has centered on the optimization of representatives of the pyridinioalkanoyl thioester (PATE) chemotype. Compounds of this type are relatively easy and inexpensive to synthesize and selectively target the zinc fingers of the nucleocapsid protein (NCp7) of HIV-1. Recently we have been exploring a new family of leads wherein the PATE reactive head structure is linked to amino acid derivatives. These ligand structures replaced the sulfonyl aromatics previously reported. Amino acid residues terminating in free carboxyl (COOH) or carboxylic acid ester (COOR) functions performed poorly in the XTT assays; however, carboxamides (CONH2) showed considerable promise. A number of other compounds with ligands based on alpha and omega amino acid derivatives have been synthesized and a library of compounds of this type is being further expanded through combinatorial substitutions at the alpha terminal nitrogen, the amide terminal nitrogen and the alpha carbon atom (amino acid side chain position). Currently, we are also preparing and exploring variants of the reactive head group structure by varying the length of the alkanoyl chain lying between the pyridine ring and the thiobenzamide group. Studies of the mechanism of zinc ejection by PATE and of NCp7 structure and function have been actively pursued. In a recent study fluorescence and electrospray ionization-mass spectrometry were used to probe the mechanism of inactivation of NCp7 by pyridinioalkanoyl thioesters. Although these compounds are shown to have potent antiviral properties in cell and target based (XTT) assays, they failed to eject zinc rapidly from NCp7. However, upon ?activation? by silver ions or, to a lesser extent by Fe2+, they reacted with NCp7 resulting in zinc ejection from both fingers. Zinc ejection followed a two-step mechanism in which the carboxyl terminal zinc is ejected faster than the amino terminal zinc as monitored by comparing the rates of zinc ejection from the whole protein and synthetic peptides corresponding to the two fingers. Both compounds covalently modified the protein with the pyridinioalkanoyl group forming thioester linkages with the cysteines of the protein via a transacylation reaction. Sequencing peptides by tandem mass spectrometry (MS/MS) from a clostripain digest of NCp7, revealed the presence of ions consistent with modifications at Cys-36 and Cys-49 of the carboxyl-terminal zinc finger. Therefore, the thioesters attack Zn finger cysteines through formation of another thioester linkage via S to S acyl transfer, a mechanism distinctly different from the action of disulfides and benzisothiazolones. In the case of disulfides, both fingers are directly attacked, followed by formation of intramolecular disulfide bonds, whereas in the case of PATEs the release of zinc from the amino-terminal finger is a consequence of structural collapse rather than direct attack by a PATE. This step brings about the desired disruption of NCp7 and its selective interaction with retroviral RNA. All the above information will be employed in molecular modeling that will include docking, molecular dynamics and molecular orbital simulations. Interpretations from these computational studies will help to direct further design, trial syntheses, and reiteration through the synthesis-screening process. - chemical interference, gel electrophoresis, nucleocapsid protein, organic synthesis, RNA synthesis, - Neither Human Subjects nor Human Tissues